Modified human growth hormone

ABSTRACT

The invention relates to the modification of human growth hormone (high) to result in human growth hormone proteins that are substantially non-immunogenic or less immunogenic than any non-modified counterpart when used in-vivo. The invention relates, furthermore, to T-cell epitome sequences deriving from high, which are immunogenic.

FIELD OF THE INVENTION

The present invention relates to polypeptides to be administeredespecially to humans and in particular for therapeutic use. Thepolypeptides are modified polypeptides whereby the modification resultsin a reduced propensity for the polypeptide to elicit an immune responseupon administration to the human subject. The invention in particularrelates to the modification of human growth hormone to result in humangrowth hormone proteins that are substantially non-immunogenic or lessimmunogenic than any non-modified counterpart when used in vivo.

BACKGROUND OF THE INVENTION

There are many instances whereby the efficacy of a therapeutic proteinis limited by an unwanted immune reaction to the therapeutic protein.Several mouse monoclonal antibodies have shown promise as therapies in anumber of human disease settings but in certain cases have failed due tothe induction of significant degrees of a human anti-murine antibody(HAMA) response [Schroff, R. W. et al (1985) Cancer Res. 45: 879-885;Shawler, D.L. et al (1985) J. Immunol. 135: 1530-1535]. For monoclonalantibodies, a number of techniques have been developed in attempt toreduce the HAMA response [WO 89/09622; EP 0239400; EP 0438310; WO91/06667]. These recombinant DNA approaches have generally reduced themouse genetic information in the final antibody construct whilstincreasing the human genetic information in the final construct.Notwithstanding, the resultant “humanized” antibodies have, in severalcases, still elicited an immune response in patients [Issacs J. D.(1990) Sem. Immunol. 2: 449, 456; Rebello, P. R. et al (1999)Transplantation 68: 1417-1420].

Antibodies are not the only class of polypeptide molecule administeredas a therapeutic agent against which an immune response may be mounted.Even proteins of human origin and with the same amino acid sequences asoccur within humans can still induce an immune response in humans.Notable examples amongst others include the therapeutic use ofgranulocyte-macrophage colony stimulating factor [Wadhwa, M. et al(1999) Clin. Cancer Res. 5: 1353-1361] and interferon alpha 2 [Russo, D.et al (1996) Bri. J. Haem. 94: 300-305; Stein, R. et al (1988) New Engl.J. Med. 318: 1409-1413]. In such situations where these human proteinsare immunogenic, there is a presumed breakage of immunological tolerancethat would otherwise have been operating in these subjects to theseproteins.

This situation is different where the human protein is beingadministered as a replacement therapy for example in a genetic diseasewhere there is a constitutional lack of the protein such as can be thecase for diseases such as hemophilia A, hemophilia B, Gauchers diseaseand numerous other examples. In such cases, the therapeutic replacementprotein may function immunologically as a foreign molecule from theoutset, and where the individuals are able to mount an immune responseto the therapeutic, the efficacy of the therapy is likely to besignificantly compromised.

Irrespective of whether the protein therapeutic is seen by the hostimmune system as a foreign molecule, or if an existing tolerance to themolecule is overcome, the mechanism of immune reactivity to the proteinis the same. Key to the induction of an immune response is the presencewithin the protein of peptides that can stimulate the activity ofT-cells via presentation on MHC class II molecules, so-called “T-cellepitopes”. Such T-cells epitopes are commonly defined as any amino acidresidue sequence with the ability to bind to MHC Class II molecules.Implicitly, a “T-cell epitope” means an epitope which when bound to MHCmolecules can be recognized by a T-cell receptor (TCR), and which can,at least in principle, cause the activation of these T-cells by engaginga TCR to promote a T-cell response.

MHC Class II molecules are a group of highly polymorphic proteins whichplay a central role in helper T-cell selection and activation. The humanleukocyte antigen group DR (HLA-DR) are the predominant isotype of thisgroup of proteins however, isotypes HLA-DQ and HLA-DP perform similarfunctions. In the human population, individuals bear two to four DRalleles, two DQ and two DP alleles. The structure of a number of DRmolecules has been solved and these appear as an open-ended peptidebinding groove with a number of hydrophobic pockets which engagehydrophobic residues (pocket residues) of the peptide [Brown et alNature (1993) 364: 33; Stern et al (1994) Nature 368: 215]. Polymorphismidentifying the different allotypes of class II molecule contributes toa wide diversity of different binding surfaces for peptides within thepeptide binding grove and at the population level ensures maximalflexibility with regard to the ability to recognise foreign proteins andmount an immune response to pathogenic organisms.

An immune response to a therapeutic protein proceeds via the MHC classII peptide presentation pathway. Here exogenous proteins are engulfedand processed for presentation in association with MHC class IImolecules of the DR, DQ or DP type. MHC Class II molecules are expressedby professional antigen presenting cells (APCs), such as macrophages anddendritic cells amongst others. Engagement of a MHC class II peptidecomplex by a cognate T-cell receptor on the surface of the T-cell,together with the cross-binding of certain other co-receptors such asthe CD4 molecule, can induce an activated state within the T-cell.Activation leads to the release of cytokines further activating otherlymphocytes such as B cells to produce antibodies or activating T killercells as a full cellular immune response.

T-cell epitope identification is the first step to epitope elimination,however there are few clear cases in the art where epitopeidentification and epitope removal are integrated into a single scheme.Thus WO98/52976 and WO00/34317 teach computational threading approachesto identifying polypeptide sequences with the potential to bind asub-set of human MHC class II DR allotypes. In these teachings,predicted T-cell epitopes are removed by the use of judicious amino acidsubstitution within the protein of interest. However with this schemeand other computationally based procedures for epitope identification[Godkin, A. J. et al (1998) J. Immunol. 161: 850-858; Sturniolo, T. etal (1999) Nat. Biotechnol. 17: 555-561], peptides predicted to be ableto bind MHC class II molecules may not function as T-cell epitopes inall situations, particularly, in vivo due to the processing pathways orother phenomena.

Equally, in vitro methods for measuring the ability of syntheticpeptides to bind MHC class II molecules, for example using B-cell linesof defined MHC allotype as a source of MHC class II binding surface andmay be applied to MHC class II ligand identification [Marshall K. W. etal. (1994) J. Immunol 152:4946-4956; O'Sullivan et al (1990) J. Immunol.145: 1799-1808; Robadey C. et al (1997) J. Immunol 159: 3238-3246].However, such techniques are not adapted for the screening multiplepotential epitopes to a wide diversity of MHC allotypes, nor can theyconfirm the ability of a binding peptide to function as a T-cellepitope.

Recently techniques exploiting soluble complexes of recombinant MHCmolecules in combination with synthetic peptides have come into use[Kern, F. et al (1998) Nature Medicine 4:975-978; Kwok, W. W. et al(2001) TRENDS in Immunol. 22:583-588]. These reagents and procedures areused to identify the presence of T-cell clones from peripheral bloodsamples from human or experimental animal subjects that are able to bindparticular MHC-peptide complexes and are not adapted for the screeningmultiple potential epitopes to a wide diversity of MHC allotypes.

Biological assays of T-cell activation provide a practical option toproviding a reading of the ability of a test peptide/protein sequence toevoke an immune response. Examples of this kind of approach include thework of Petra et al using T-cell proliferation assays to the bacterialprotein staphylokinase, followed by epitope mapping using syntheticpeptides to stimulate T-cell lines [Petra, A. M. et al (2002) J.Immunol. 168: 155-161]. Similarly, T-cell proliferation assays usingsynthetic peptides of the tetanus toxin protein have resulted indefinition of immunodominant epitope regions of the toxin [Reece J. C.et al (1993) J. Immunol. 151: 6175-6184]. WO99/53038 discloses anapproach whereby T-cell epitopes in a test protein may be determinedusing isolated sub-sets of human immune cells, promoting theirdifferentiation in vitro and culture of the cells in the presence ofsynthetic peptides of interest and measurement of any inducedproliferation in the cultured T-cells. The same technique is alsodescribed by Stickler et al [Stickler, M. M. et al (2000) J.Immunotherapy 23:654-660], where in both instances the method is appliedto the detection of T-cell epitopes within bacterial subtilisin. Such atechnique requires careful application of cell isolation techniques andcell culture with multiple cytoline supplements to obtain the desiredimmune cell sub-sets (dendritic cells, CD4+and or CD8+ T-cells) and isnot conducive to rapid through-put screening using multiple donorsamples.

As depicted above and as consequence thereof, it would be desirable toidentify and to remove or at least to reduce T-cell epitopes from agiven in principal therapeutically valuable but originally immunogenicpeptide, polypeptide or protein. One of these potential therapeuticallyvaluable molecules is human growth hormone (herein abbreviated to hGH).

Natural hGH is a pituitary hormone of 22 kDa molecular weight and 191amino acid residues. An alternative 20 kDa product derived byalternative splicing is also recognised and has some altered propertiescompared to the 22 kDa form [Wada, M. et al (1997) Mol. Cell Endocrinol.133: 99-107]. The 22 kDa protein been produced using recombinanttechniques in a variety of host organisms including E.coli [Goeddel, D.et al (1979) Nature 281: 544-548] Bacillus subtilis [Honjo, J. et al(1987) J. Biotech 6: 191-204], yeast [Hiramatsu, R. et al (1991) Appl.Environ. Microbiol. 57: 2052-2056] and animal cells [Lupker, J. et al(1983) Gene 24: 281-287]. Pharmaceutical preparations of hGH are usedfor the treatment of pituitary dwarfism, paediatric chronic renalfailure and similar indications. In addition to its ability to promotegrowth, the protein has a variety of biological activities includingactivation of macrophages and insulin like effects [Chawler, R. (1993)Ann. Rev. Med. 34: 519; Edwards, C. et al (1988) Science 239: 769].

The present invention is concerned with human growth hormone (hGH) andthe amino acid sequence of the secreted form of the hGH protein depictedin single-letter code is as follows:

-   FPTIPLSRLFQNAMLRAHRLHQLAFDTYEEFEEAYIPKEQKYSFLQAPQASLCFSESIPTPSNRE    QAQQKSNLQLLRISLLLIQSWLEPVGFLRSVFANSLVYGASDSDVYDLLKDLEEGIQTLMGRLED    GSPRTGQAFKQTYAKFDANSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

It is a particular objective of the present invention to providemodified hGH proteins in which the immune characteristic is modified bymeans of reduced numbers of potential T-cell epitopes.

Others have provided hGH molecules including modified hGH and schemesfor its recombinant production, purification and therapeutic use [EP0107890, U.S. Pat. No. 4,517,181, EP 0105759; U.S. Pat. No. 4,703,035;U.S. Pat. No. 4,658,021; EP0022242; EP0001929; EP0001939; U.S. Pat. No.4,342,832; U.S. Pat. No. 4,601,980; U.S. Pat. No. 4,604,359; U.S. Pat.No. 4,634,677; U.S. Pat. No. 4,898,830; U.S. Pat. No. 5,424,119; U.S.Pat. No. 4,366,246; U.S. Pat. No. 4,425,437; U.S. Pat. No. 4,431,739;U.S. Pat No. 4,563,424; U.S. Pat. No. 4,571,421; EP 0131843; EP 0319049;U.S. Pat. No. 4,831,120; U.S. Pat. No. 4,871,835; U.S. Pat. No.4,997,916; U.S. Pat. No. 5,612,315; U.S. Pat. No.5,633,352; U.S. Pat.No. 5,618,697; U.S. Pat. No. 5,635,604; EP 0127658; EP 0217814; U.S.Pat. No. 5,898,030; EP0804223] but these teachings do not address theimportance of T cell epitopes to the immunogenic properties of theprotein nor have been conceived to directly influence said properties ina specific and controlled way according to the scheme of the presentinvention. An example in this regard is provided by Lowman and Wells[Lowman H. B. & Wells J. A. (1993) J. Mol. Biol. 243: 564-578] who haveused phage display in the creation of a hGH variant which exhibits anapproximately 400-fold increased binding affinity for the hGH receptor.This high affinity variant contains fifteen amino acid substitutions butno consideration of immunological properties of the new variant moleculehas been made.

However, as disclosed for the first time herein below, the presentinventors have discovered that of these fifteen substitutions in thehigh affinity variant, seven (at positions 10, 14, 42, 45, 54, 176 and179) can be expected to provide immunological benefit according to thescheme of the present invention.

It is highly desired to provide hGH with reduced or absent potential toinduce an immune response in the human subject.

SUMMARY AND DESCRIPTION OF THE INVENTION

The present invention provides for modified forms of hGH, in which theimmune characteristic is modified by means of reduced or removed numbersof potential T-cell epitopes.

The invention discloses sequences identified within the hGH primarysequence that are potential T-cell epitopes by virtue of MHC class IIbinding potential. This disclosure specifically pertains the human hGHprotein sequence given above herein and comprising 191 amino acidresidues.

The present invention discloses the major regions of the hGH primarysequence that are immunogenic in man and thereby provides the criticalinformation required to conduct modification to the sequences toeliminate or reduce the immunogenic effectiveness of these sites.

In one embodiment, synthetic peptides comprising the immunogenic regionscan be provided in pharmaceutical composition for the purpose ofpromoting a tolerogenic response to the whole molecule.

In a further embodiment hGH molecules modified within the epitoperegions herein disclosed can be used in pharmaceutical compositions.

In summary the invention relates to the following issues:

-   -   a modified molecule having the biological activity of hGH and        being substantially non-immunogenic or less immunogenic than any        non-modified molecule having the same biological activity when        used in vivo;    -   an accordingly specified molecule, wherein said loss of        immunogenicity is achieved by removing one or more T-cell        epitopes derived from the originally non-modified molecule;    -   an accordingly specified molecule, wherein said loss of        immunogenicity is achieved by reduction in numbers of MHC        allotypes able to bind peptides derived from said molecule;    -   an accordingly specified molecule, wherein one T-cell epitope is        removed;    -   an accordingly specified molecule, wherein said originally        present T-cell epitopes are MHC class II ligands or peptide        sequences which show the ability to stimulate or bind T-cells        via presentation on class II;    -   an accordingly specified molecule, wherein said peptide        sequences are selected from the group as depicted in Table 1;    -   an accordingly specified molecule, wherein 1-9 amino acid        residues preferably one amino acid residue in any of the        originally present T-cell epitopes are altered;    -   an accordingly specified molecule, wherein the alteration of the        amino acid residues is substitution, addition or deletion of        originally present amino acid(s) residue(s) by other amino acid        residue(s) at specific position(s);    -   an accordingly specified molecule, wherein one or more of the        amino acid residue substitutions are carried out as indicated in        Table 2;    -   an accordingly specified molecule, wherein (additionally) one or        more of the amino acid residue substitutions are carried out as        indicated in Table 3 for the reduction in the number of MHC        allotypes able to bind peptides derived from said molecule;    -   an accordingly specified molecule, wherein, if necessary,        additionally further alteration usually by substitution,        addition or deletion of specific amino acid(s) is conducted to        restore biological activity of said molecule;    -   an accordingly specified hGH molecule, wherein one or more of        the amino acid substitutions is conducted at a position        corresponding to any of the amino acids specified within Tables        2 or 3;    -   an accordingly specified hGH molecule, wherein one or more of        the amino acid substitutions is conducted at a position        corresponding to any of the amino acids specified within Tables        2 or 3 but excluding any of those substitutions known from the        record of hGH genetic mutations to be incompatible with        functional protein;    -   a pharmaceutical composition comprising any of the peptides or        modified peptides of above having the activity of binding to MHC        class II;    -   a DNA sequence or molecule which codes for any of said specified        modified molecules as defined above and below;    -   a pharmaceutical composition comprising a modified molecule        having the biological activity of hGH as defined above and/or in        the claims, optionally together with a pharmaceutically        acceptable carrier, diluent or excipient;    -   a method for manufacturing a modified molecule having the        biological activity of hGH as defined in any of the claims of        the above-cited claims comprising the following steps: (i)        determining the amino acid sequence of the polypeptide or part        thereof; (ii) identifying one or more potential T-cell epitopes        within the amino acid sequence of the protein by any method        including determination of the binding of the peptides to MHC        molecules using in vitro or in silico techniques or biological        assays; (iii) designing new sequence variants with one or more        amino acids within the identified potential T-cell epitopes        modified in such a way to substantially reduce or eliminate the        activity of the T-cell epitope as determined by the binding of        the peptides to MHC molecules using in vitro or in silico        techniques or biological assays; (iv) constructing such sequence        variants by recombinant DNA techniques and testing said variants        in order to identity one or more variants with desirable        properties; and (v) optionally repeating steps (ii)-(iv);    -   an accordingly specified method, wherein step (iii) is carried        out by substitution, addition or deletion of 1-9 amino acid        residues in any of the originally present T-cell epitopes;    -   an accordingly specified method, wherein the alteration is made        with reference to an homologous protein sequence and/or in        silico modeling techniques;    -   an accordingly specified method, wherein step (ii) of above is        carried out by the following steps: (a) selecting a region of        the peptide having a known amino acid residue sequence; (b)        sequentially sampling overlapping amino acid residue segments of        predetermined uniform size and constituted by at least three        amino acid residues from the selected region; (c) calculating        MHC Class II molecule binding score for each said sampled        segment by summing assigned values for each hydrophobic amino        acid residue side chain present in said sampled amino acid        residue segment; and (d) identifying at least one of said        segments suitable for modification, based on the calculated MHC        Class II molecule binding score for that segment, to change        overall MHC Class II binding score for the peptide without        substantially reducing therapeutic utility of the peptide;        step (c) is preferably carried out by using a Böhm scoring        function modified to include 12-6 van der Waal's ligand-protein        energy repulsive term and ligand conformational energy term        by (1) providing a first data base of MHC Class II molecule        models; (2) providing a second data base of allowed peptide        backbones for said MHC Class II molecule models; (3) selecting a        model from said first data base; (4) selecting an allowed        peptide backbone from said second data base; (5) identifying        amino acid residue side chains present in each sampled        segment; (6) determining the binding affinity value for all side        chains present in each sampled segment; and repeating steps (1)        through (5) for each said model and each said backbone;    -   a 13mer T-cell epitope peptide having a potential MHC class II        binding activity and created from non-modified hGH, selected        from the group as depicted in Table 1 and its use for the        manufacture of hGH having substantially no or less        immunogenicity than any non-modified molecule with the same        biological activity when used in vivo;    -   a peptide sequence consisting of at least 9 consecutive amino        acid residues of a 13mer T-cell epitope peptide as specified        above and its use for the manufacture of hGH having        substantially no or less immunogenicity than any non-modified        molecule with the same biological activity when used in vivo;    -   using a panel of synthetic peptides in a biological T-cell assay        to map the immunogenic region(s) of human hGH;    -   using a panel of hGH protein variants in a biological T-cell        assay to select variants displaying minimal immunogenicity in        vitro;    -   using a panel of synthetic peptide variants in a biological        T-cell assay to select peptide sequences displaying minimal        immunogenicity in vitro;    -   using biological assays of T-cell stimulation to select a        protein variant which exhibits a stimulation index of less than        2.0 and preferably less than 1.8 in a naïve T-cell assay;    -   construction of a T-cell epitope map of hGH protein using PBMC        isolated from healthy donors and a screening method involving        the steps comprising: i) antigen priming in vitro using        synthetic peptide or whole protein immunogen for a culture        period of up to 7 days; ii) addition of IL-2 and culture for up        to 3 days; iii) addition of primed T cells to autologous        irradiated PBMC and re-challenge with antigen for a further        culture period of 4 days and iv) measurement of proliferation        index by any suitable method;    -   hGH derived peptide sequences able to evoke a stimulation index        of greater than 1.8 and preferably greater than 2.0 in a naïve        T-cell assay;    -   hGH derived peptide sequences having a stimulation index of        greater than 1.8 and preferably greater than 2.0 in a naïve        T-cell assay wherein the peptide is modified to a minimum extent        and tested in the naïve T-cell assay and found to have a        stimulation index of less than 2.0;    -   hGH derived peptide sequences sharing 100% amino acid identity        with the wild-type protein sequence and able to evoke a        stimulation index of 1.8 or greater and preferably greater than        2.0 in a T-cell assay;    -   an accordingly specified hGH peptide sequence modified to        contain less than 100% amino acid identity with the wild-type        protein sequence and evoking a stimulation index of less than        2.0 when tested in a T-cell assay;    -   a hGH molecule containing a modified peptide sequence which when        individually tested evokes a stimulation index of less than 2.0        in a T-cell assay,    -   a hGH molecule containing modifications such that when tested in        a T-cell assay evokes a reduced stimulation index in comparison        to a non modified protein molecule;    -   a hGH molecule in which the immunogenic regions have been mapped        using a T-cell assay and then modified such that upon re-testing        in a T-cell assay the modified protein evokes a stimulation        index smaller than the parental (non-modified) molecule and most        preferably less than 2.0.

The term “T-cell epitope” means according to the understanding of thisinvention an amino acid sequence which is able to bind MHC class II,able to stimulate T-cells and/or also to bind (without necessarilymeasurably activating) T-cells in complex with MHC class II.

The term “peptide” as used herein and in the appended claims, is acompound that includes two or more amino acids. The amino acids arelinked together by a peptide bond (defined herein below). There are 20different naturally occurring amino acids involved in the biologicalproduction of peptides, and any number of them may be linked in anyorder to form a peptide chain or ring. The naturally occurring aminoacids employed in the biological production of peptides all have theL-configuration. Synthetic peptides can be prepared employingconventional synthetic methods, utilizing L-amino acids, D-amino acids,or various combinations of amino acids of the two differentconfigurations. Some peptides contain only a few amino acid units. Shortpeptides, e.g., having less than ten amino acid units, are sometimesreferred to as “oligopeptides”. Other peptides contain a large number ofamino acid residues, e.g. up to 100 or more, and are referred to as“polypeptides”. By convention, a “polypeptide” may be considered as anypeptide chain containing three or more amino acids, whereas a“oligopeptide” is usually considered as a particular type of “short”polypeptide. Thus, as used herein, it is understood that any referenceto a “polypeptide” also includes an oligopeptide. Further, any referenceto a “peptide” includes polypeptides, oligopeptides, and proteins. Eachdifferent arrangement of amino acids forms different polypeptides orproteins. The number of polypeptides—and hence the number of differentproteins—that can be formed is practically unlited. “Alpha carbon (Cα)”is the carbon atom of the carbon-hydrogen (CH) component that is in thepeptide chain. A “side chain” is a pendant group to Cα that can comprisea simple or complex group or moiety, having physical dimensions that canvary significantly compared to the dimensions of the peptide.

The invention may be applied to any hGH species of molecule withsubstantially the same primary amino acid sequences as that disclosedherein and would include therefore hGH molecules derived by geneticengineering means or other processes and may contain more or less than191 amino acid residues. Many of the peptide sequences of the presentdisclosure are in common with peptide sequences derived from hGHproteins of non-human origin or are at least substantially the same asthose from non-human hGH proteins. Such protein sequences equallytherefore fall under the scope of the present invention.

The invention is conceived to overcome the practical reality thatsoluble proteins introduced with therapeutic intent in man trigger animmune response resulting in development of host antibodies that bind tothe soluble protein. The present invention seeks to address this byproviding hGH proteins with altered propensity to elicit an immuneresponse on administration to the human host. According to the methodsdescribed herein, the inventors have discovered the regions of the hGHmolecule comprising the critical T-cell epitopes driving the immuneresponses to this protein.

The general method of the present invention leading to the modified hGHcomprises the following steps:

-   -   (a) determining the amino acid sequence of the polypeptide or        part thereof;    -   (b) identifying one or more potential T-cell epitopes within the        amino acid sequence of the protein by any method including        determination of the binding of the peptides to MHC molecules        using in vitro or in silico techniques or biological assays;    -   (c) designing new sequence variants with one or more amino acids        within the identified potential T-cell epitopes modified in such        a way to substantially reduce or eliminate the activity of the        T-cell epitope as determined by the binding of the peptides to        MHC molecules using in vitro or in silico techniques or        biological assays. Such sequence variants are created in such a        way to avoid creation of new potential T-cell epitopes by the        sequence variations unless such new potential T-cell epitopes        are, in turn, modified in such a way to substantially reduce or        eliminate the activity of the T-cell epitope; and    -   (d) constructing such sequence variants by recombinant DNA        techniques and testing said variants in order to identify one or        more variants with desirable properties according to well known        recombinant techniques.

The identification of potential T-cell epitopes according to step (b)can be carried out according to methods describes previously in the art.Suitable methods are disclosed in WO 98/59244; WO 98/52976; WO 00/34317and may preferably be used to identify binding propensity of hGH-derivedpeptides to an MHC class II molecule.

Another very efficacious method for identifying T-cell epitopes bycalculation is described in the Example 1 which is a preferredembodiment according to this invention.

The results of an analysis according to step (b) of the above scheme andpertaining to the human hGH protein sequence is presented in Table 1.TABLE 1 Peptide sequences in human hGH with potential human MHC class IIbinding activity. PTIPLSRLFQNAM, IPLSRLFQNAMLR, SRLFQNAMLRAHR,RLFQNAMLRAHRL, NAMLRAHRLHQLA, AMLRAHRLRQLAF, HRLHQLAFDTYEE,HQLAFDTYEEFEE, LAFDTYEEFEEAY, DTYEEFEEAYIPK, EEFEEAYIPKEQK,EAYIPKEQKYSFL, AYIPKEQKYSFLQ, QKYSFLQAPQASL, YSFLQAPQASLCF,SFLQAPQASLCFS, ASLCFSESIPTPS, LCFSESIPTPSNR, ESIPTPSNREQAQ,SNLQLLRISLLLI, LQLLRISLLLIQS, QLLRISLLLIQSW, LRISLLLIQSWLE,ISLLLIQSWLEPV, SLLLIQSWLEPVG, LLLIQSWLEPVGF, LLIQSWLEPVGFL,QSWLEPVGFLRSV, SWLEPVGFLRSVF, EPVGFLRSVFANS, VGFLRSVFANSLV,GFLRSVFANSLVY, RSVFANSLVYGAS, SVFANSLVYGASD, NSLVYGASDSDVY,SLVYGASDSDVYD, LVYGASDSDVYDL, SDVYDLLKDLEEG, DVYDLLKDLEEGI,YDLLKDLEEGIQT, DLLKDLEEGIQTL, KDLEEGIQTLMGR, EGIQTLMGRLEDG,QTLMGRLEDGSPR, TLMGRLEDGSPRT, GRLEDGSPRTGQA, QAFKQTYAKFDAN,QTYAKFDANSHND, AKFDANSHNDDAL, DALLKNYGLLYCF, ALLKNYGLLYCFR,KNYGLLYCFRKDM, YGLLYCFRKDMDK, GLLYCFRKDMDKV, LLYCFRKDMDKVE,YCFRKDMDKVETF, KDMDKVETFLRIV, DKVETFLRIVQCR, ETFLRIVQCRSVE,TFLRIVQCRSVEG, LRIVQCRSVEGSC, RIVQCRSVEGSCG

Peptides are 13mers, amino acid are identified using single lettercodes.

The results of a design and constructs according to step (c) and (d) ofthe above scheme and pertaining to the modified molecule of thisinvention is presented in Tables 2 and 3. TABLE 2 Substitutions leadingto the elimination of T-cell epitopes of human hGH (WT = wild typeresidue). Residue WT # Residue Substitution 4 I A C D E G H K N P Q R ST 6 L A C D E G H K N P Q R S T 9 L A C D E G H K N P Q R S T 10 F A C DE G H K N P Q R S T 14 M A C D E G H K N P Q R S T 15 L A C D E G H K NP Q R S T 20 L A C D E G H K N P Q R S T 23 L A C D E G H K N P Q R S T25 F A C D E G H K N P Q R S T 28 Y A C D E G H K N P Q R S T 31 F A C DE G H K N P Q R S T 35 Y A C D E G H K N P Q R S T 36 I A C D E G H K NP Q R S T 42 Y A C D E G H K N P Q R S T 44 F A C D E G H K N P Q R S T45 L A C D E G H K N P Q R S T 52 L A C D E G H K N P Q R S T 54 F A C DE G H K N P Q R S T 58 I A C D E G H K N P Q R S T 73 L A C D E G H K NP Q R S T 75 L A C D E G H K N P Q R S T 76 L A C D E G H K N P Q R S T78 I A C D E G H K N P Q R S T 80 L A C D E G H K N P Q R S T 81 L A C DE G H K N P Q R S T 82 L A C D E G H K N P Q R S T 83 I A C D E G H K NP Q R S T 86 W A C D E G H K N P Q R S T 87 L A C D E G H K N P Q R S T90 V A C D E G H K N P Q R S T 92 F A C D E G H K N P Q R S T 93 L A C DE G H K N P Q R S T 96 V A C D E G H K N P Q R S T 97 F A C D E G H K NP Q R S T 101 L A C D E G H K N P Q R S T 102 V A C D E G H K N P Q R ST 103 Y A C D E G H K N P Q R S T 110 V A C D E G H K N P Q R S T 111 YA C D E G H K N P Q R S T 113 L A C D E G H K N P Q R S T 114 L A C D EG H K N P Q R S T 117 L A C D E G H K N P Q R S T 121 I A C D E G H K NP Q R S T 124 L A C D E G H K N P Q R S T 125 M A C D E G H K N P Q R ST 128 L A C D E G H K N P Q R S T 139 F A C D E G H K N P Q R S T 143 YA C D E G H K N P Q R S T 146 F A C D E G H K N P Q R S T 156 L A C D EG H K N P Q R S T 157 L A C D E G H K N P Q R S T 160 Y A C D E G H K NP Q R S T 162 L A C D E G H K N P Q R S T 163 L A C D E G H K N P Q R ST 164 Y A C D E G H K N P Q R S T 166 F A C D E G H K N P Q R S T 170 MA C D E G H K N P Q R S T 173 V A C D E G H K N P Q R S T 176 F A C D EG H K N P Q R S T 177 L A C D E G H K N P Q R S T 179 I A C D E G H K NP Q R S T 180 V A C D E G H K N P Q R S T

TABLE 3 Additional substitutions leading to the removal of a potentialT-cell epitope for 1 or more MHC allotypes. WT Residue ResidueSubstitution 6 L M W Y 9 L I M V W Y 11 Q H 12 N A C G P T 14 M F I V WY 15 L F I M V W Y 16 R A C G P 17 A D E H K N P Q R S T 18 H P 19 R A CG P 20 L F I M V W Y 21 H P T 22 Q A C G P T 23 L W I M V W Y 25 F W Y26 D P T 29 E T 30 E H 31 F I V W 33 E H T 34 A I P T Y 36 I F W Y 39 ET 44 F W Y 45 L F I M W Y 47 A C D E G H K N P Q R S T 49 Q D H 50 A D EH K N P Q R S T 51 S T 52 L F I M V W Y 53 C H P T 73 L F I M V W Y 74 QA C G P 75 L F I M V W Y 76 L F I M V W Y 77 R A C G P 78 I W Y 79 S A CG P T 80 L F I M V W Y 81 L F I M V W Y 82 L F I M V W Y 83 I F W Y 84 QA C G P T 85 S A C G P 86 W A C D E G H K N P Q R S T 87 L A C D E G H KM N P Q R S T W Y 88 E P T 90 V M W Y 93 L F I M W Y 94 R A C G P 95 S AC G P T 96 V F I M W Y 97 F M W Y 98 A D E H K N P Q R S T 99 N A C G HP T 100 S A C G P T 101 L F I M V W Y 102 V F I M W Y 104 G D E H K N PQ R S T 105 A C D E H K N P Q R S T 106 S A C D G H P 107 D A C G P T108 S A C G P T 109 D A C G H P T 110 V M W Y 112 D A C G P 113 L F I MV W Y 114 L F I M V W Y 115 K A C G H P 116 D A C G H P T 117 L F I M WY 118 E A C G H P T 119 E H P T 120 G D E H K N P Q R S T 121 I M W Y122 Q A C G H P T 123 T A C G P 124 L F I V W Y 125 M F I V W Y 126 G DE H K N P Q S 127 R A C G P T 128 L F I M V W Y 129 E A C G P T 130 D HP 131 G C D E H K N P Q R S T 132 S A C G P 133 P T 134 R P T 135 T A CG P 136 G H P T 139 F M W Y 140 K A C G P 141 Q A C G P 142 T P 143 Y W144 A D E H K N P Q R S T 145 K P T 147 D P T 148 A D E H K N P Q R S T149 N A C G P T 151 H P T 156 L F I M V W Y 157 L F I M W Y 158 K A C GP 159 N A C G H P T 161 G D E H K N P Q S 162 L F I M V W Y 163 L F I VW Y 165 C D E H K N P Q R S T 167 R A C G H P 168 K H P S T 169 D A C GP 170 M I V W Y 171 D A C G P 173 V M W Y 175 T H 176 F W Y 177 L I M WY 178 R H P T 179 I W Y 180 V F I M W Y 181 Q A C G P 182 C F H L P T WY 183 R I P T V Y 184 S A C D F G H I L M P T V W Y 185 V A C D E F G HI K L M N P Q R S T W Y 186 E I P T Y 187 G F H I P T V W Y 188 S F I PT V W Y

A further technical approach to the detection of T-cell epitopes is viabiological T-cell assay. For the detection of T-cell epitopes within thehGH molecule a particularly effective method would be to test all or anyof the peptide sequences of Table 1 for their ability to evoke anproliferative response in human T-cells cultured in vitro. The preferredmethod would be to exploit peripheral blood mononuclear cells (PBMC)from individuals where, in effect, the hGH protein antigen due to thenature of the genetic deficit in the individuals may constitute aforeign protein. In this sense, the protein is most likely to representa potent antigen in vivo. This can be achieved using T cells subjectedto several rounds of antigen (hGH) stimulation in vitro followedimmediately by expansion in the presence of IL-2. For establishingpolyclonal T cell lines 2-3 rounds of antigen stimulation are generallysufficient to generate a large number of antigen specific cells. Theseare used to screen large numbers of synthetic peptides (for example inthe form of peptide pools), and they may be cryogenically stored to beused at a later date. After the initial round of antigen stimulationcomprising co-incubation of the hGH antigen and PBMC for 7 dayssubsequent re-challenges with antigen are performed in the presence ofmost preferably autologous irradiated PBMC as antigen presenting cells.These rounds of antigen selection are performed for 3-4 days and areinterspersed by expansion phases comprising stimulation with IL-2 whichmay be added every 3 days for a total period of around 9 days. The finalre-challenge is performed using T-cells that have been “rested”, that isT cells which have not been IL-2 stimulated for around 4 days. Thesecells are stimulated with antigen (e.g. synthetic peptide or wholeprotein) using most preferably autologous antigen presenting cells aspreviously for around 4 days and the subsequent proliferative response(if any) is measured thereafter. The proliferative response can bemeasured by any convenient means and a widely known method for examplewould be to use an ³H-thymidine incorporation assay.

Accordingly the method embodied herein above comprises the production ofT-cell lines or oligoclonal cultures derived from PBMC samples takenfrom individuals in whom previous therapeutic replacement therapy withhGH has been initiated to and in whom the replacement therapy hasresulted in the induction of an immune response to the therapeuticprotein. The lines or cultures from such individuals, are contacted withpreparations of synthetic peptides or whole proteins and any in vitrothe proliferative effects are measured. For any of the individualsynthetic peptides or proteins, variants may be produced and re-testedfor a continued ability to promote a significant proliferative responsein the T-cell lines or cultures. Thus for example synthetic peptidescontaining any of the substitutions or combination of substitutionsidentified in Table 2 or Table 3 may be tested in such an assay.

Under this scheme it could be expected that the epitope map of the thehGH protein defined by the T-cell repertoire of a significant number ofthese individuals will be representative of the most prevalent peptideepitopes that are capable of presentation in the in vivo context. Inthis sense, PBMC from patients in whom there is a previouslydemonstrated immune response constitute the products of an in vivopriming step and given that the use of PBMC cell lines from suchindividuals is in principle an immunological in vitro recall assay, itfurther provides the practical benefit of there being the capacity for amuch larger magnitude of proliferative response to any given stimulatingpeptide or protein. This reduces the technical challenge of conducting aproliferation measurement and in such a situation may give theopportunity for definition of a possible hierarchy of immunodominantepitopes as is the case for hGH which is demonstrated hereincomputationally to harbour multiple MHC class II peptide ligands andtherefore multiple or complex (i.e. overlapping) T-cell epitopes.

Whilst it is particularly useful to establish T-cell lines ofoligoclonal cultures from individuals in whom previous therapeutic hGHreplacement therapy has resulted in the induction of an immune responseto hGH, these are not the only source of cells which can be used to mapthe in vivo related immunogenic epitopes. Assay of naïve T-cells takenfrom healthy donors can equally be used, however in such an instance themagnitude of the stimulation index scored for any individual peptide islikely to be low requiring sensitive measurement to discern the peptideor protein induced stimulation from that of the background. Theinventors have established in the operation of such an assay using wellknown techniques that a stimulation index equal to or greater than 2.0is a useful measure of induced proliferation where the stimulation indexis derived by division of the proliferation score measured (e.g. countsper minute if using ³H-thymidine incorporation) to the test (poly)peptide by the proliferation score measured in cells not contacted witha test (poly)peptide. A suitable method of this type is detailed inExample 2.

Where multiple potential epitopes are identified and in particular wherea number of peptide sequences are found to be able to stimulate T-cellsin a biological assay, cognisance may also be made of the structuralfeatures of the protein in relation to its propensity to evoke an immuneresponse via the MHC class II presentation pathway. For example wherethe crystal structure of the protein of interest is known thecrystallographic B-factor score may be analysed for evidence ofstructural disorder within the protein, a parameter suggested tocorrelate with the proximity to the biologically relevant immunodominantpeptide epitopes [Dai G. et al (2001) J. Biological Chem. 276:41913-41920]. Such an analysis when conducted on the hGH crystalstructures [PDB ID:1 HGU Chantalat, L. et al (1995), Protein And PeptideLetters 2:333 & PDB ID 1A22 Clackson, T. et al (1998), J. Mol. Biol.277: 1111] suggests a high likelihood for multiple immunodominantepitopes with at least 7 peaks of above mean B-factor scores within thenon-receptor bound structure [PDB ID 1HGU]. This analysis indicates thatthe biologically relevant T-cell epitopes map to regions in the hGHsequence downstream from glutamine residue 41. Accordingly, under thescheme of the present; of the amino acid substitutions listed in Table 2and Table 3, the most preferred substitutions comprise those directed toresidues encompassed within residue numbers 42-180.

In practice a number of variant hGH proteins will be produced and testedfor the desired immune and functional characteristic. Reference can bemade to the mutations in the published literature known to result inalteration of the functional characteristics of the molecule [Lowman H.B. & Wells J. A. (1993) J. Mol. Biol. 243: 564-578; Wells J. A. et al(1993) Recent Prog. Horm. Res. 48: 253-275] and those substitutionslisted in Table 2 and Table 3 which are also known to be deleterious tothe protein function may be excluded for analysis or alternativelycompensatory mutation may be conducted in order to restore functionalactivity of the protein. In all instances the variant proteins will mostpreferably be produced by the widely known methods of recombinant DNAtechnology although other procedures including chemical synthesis of hGHfragments may be contemplated. The invention relates to hGH analogues inwhich substitutions of at least one amino acid residue have been made atpositions resulting in a substantial reduction in activity of orelimination of one or more potential T-cell epitopes from the protein.It is most preferred to provide hGH molecules in which amino acidmodification (e.g. a substitution) is conducted within the mostimmunogenic regions of the parent molecule. The major preferredembodiments of the present invention comprise hGH molecules for whichany of the MHC class II ligands are altered such as to eliminate bindingor otherwise reduce the numbers of MHC allotypes to which the peptidecan bind.

For the elimination of T-cell epitopes, amino acid substitutions arepreferably made at appropriate points within the peptide sequencepredicted to achieve substantial reduction or elimination of theactivity of the T-cell epitope. In practice an appropriate point willpreferably equate to an amino acid residue binding within one of thepockets provided within the MHC class II binding groove.

It is most preferred to alter binding within the first pocket of thecleft at the so-called P1 or P1 anchor position of the peptide. Thequality of binding interaction between the P1 anchor residue of thepeptide and the first pocket of the MHC class II binding groove isrecognized as being a major determinant of overall binding affinity forthe whole peptide. An appropriate substitution at this position of thepeptide will be for a residue less readily accommodated within thepocket, for example, substitution to a more hydrophilic residue. Aminoacid residues in the peptide at positions equating to binding withinother pocket regions within the MHC binding cleft are also consideredand fall under the scope of the present.

It is understood that single amino acid substitutions within a givenpotential T-cell epitope are the most preferred route by which theepitope may be eliminated. Combinations of substitution within a singleepitope may be contemplated and for example can be particularlyappropriate where individually defined epitopes are in overlap with eachother. Moreover, amino acid substitutions either singly within a givenepitope or in combination within a single epitope may be made atpositions not equating to the “pocket residues” with respect to the MHCclass II binding groove, but at any point within the peptide sequence.Substitutions may be made with reference to an homologues structure orstructural method produced using in silico techniques known in the artand may be based on known structural features of the molecule accordingto this invention. All such substitutions fall within the scope of thepresent invention.

Amino acid substitutions other than within the peptides identifiedherein may be contemplated particularly when made in combination withsubstitution(s) made within a listed peptide. For example a change maybe contemplated to restore structure or biological activity of thevariant molecule. Such compensatory changes and changes to includedeletion or addition of particular amino acid residues from the hGHpolypeptide resulting in a variant with desired activity and incombination with changes in any of the disclosed peptides fall under thescope of the present.

In as far as this invention relates to modified hGH, compositionscontaining such modified hGH proteins or fragments of modified hGHproteins and related compositions should be considered within the scopeof the invention. In another aspect, the present invention relates tonucleic acids encoding modified hGH entities. In a further aspect thepresent invention relates to methods for therapeutic treatment of humansusing the modified hGH proteins.

In a further aspect still, the invention relates to methods fortherapeutic treatment using pharmaceutical preparations comprisingpeptide or derivative molecules with sequence identity or part identitywith the sequences herein disclosed.

EXAMPLE 1

There are a number of factors that play important roles in determiningthe total structure of a protein or polypeptide. First, the peptidebond, i.e., that bond which joins the amino acids in the chain together,is a covalent bond. This bond is planar in structure, essentially asubstituted amide. An “amide” is any of a group of organic compoundscontaining the grouping —CONH—.

The planar peptide bond linking Cα of adjacent amino acids may berepresented as depicted below:

Because the O═C and the C—N atoms lie in a relatively rigid plane, freerotation does not occur about these axes. Hence, a plane schematicallydepicted by the interrupted line is sometimes referred to as an “amide”or “peptide plane” plane wherein lie the oxygen (O), carbon (C),nitrogen (N), and hydrogen (H) atoms of the peptide backbone. Atopposite corners of this amide plane are located the Cα atoms. Sincethere is substantially no rotation about the O═C and C—N atoms in thepeptide or amide plane, a polypeptide chain thus comprises a series ofplanar peptide linkages joining the Cα atoms. A second factor that playsan important role in defining the total structure or conformation of apolypeptide or protein is the angle of rotation of each amide planeabout the common Cα linkage. The terms “angle of rotation” and “torsionangle” are hereinafter regarded as equivalent terms. Assuming that theO, C, N, and H atoms remain in the amide plane (which is usually a validassumption, although there may be some slight deviations from planarityof these atoms for some conformations), these angles of rotation definethe N and R polypeptide's backbone conformation, i.e., the structure asit exists between adjacent residues. These two angles are known as φ andψ. A set of the angles φ₁, ψ₁, where the subscript i represents aparticular residue of a polypeptide chain, thus effectively defines thepolypeptide secondary structure. The conventions used in defining the φ,ψ angles, i.e., the reference points at which the amide planes form azero degree angle, and the definition of which angle is φ, and whichangle is ψ, for a given polypeptide, are defined in the literature. See,e.g,, Ramachandran et al. Adv. Prot. Chem. 23:283-437 (1968), at pages285-94, which pages are incorporated herein by reference.

The present method can be applied to any protein, and is based in partupon the discovery that in humans the primary Pocket 1 anchor positionof MHC Class II molecule binding grooves has a well designed specificityfor particular amino acid side chains. The specificity of this pocket isdetermined by the identity of the amino acid at position 86 of the betachain of the MHC Class II molecule. This site is located at the bottomof Pocket 1 and determines the size of the side chain that can beaccommodated by this pocket.

Marshall, K. W., J. Immunol., 152:4946-4956 (1994). If this residue is aglycine, then all hydrophobic aliphatic and aromatic amino acids(hydrophobic aliphatics being: valine, leucine, isoleucine, methionineand aromatics being: phenylalanine, tyrosine and tryptophan) can beaccommodated in the pocket, a preference being for the aromatic sidechains. If this pocket residue is a valine, then the side chain of thisamino acid protrudes into the pocket and restricts the size of peptideside chains that can be accommodated such that only hydrophobicaliphatic side chains can be accommodated. Therefore, in an amino acidresidue sequence, wherever an amino acid with a hydrophobic aliphatic oraromatic side chain is found, there is the potential for a MHC Class IIrestricted T-cell epitope to be present. If the side-chain ishydrophobic aliphatic, however, it is approximately twice as likely tobe associated with a T-cell epitope than an aromatic side chain(assuming an approximately even distribution of Pocket 1 typesthroughout the global population).

A computational method embodying the present invention profiles thelikelihood of peptide regions to contain T-cell epitopes as follows:

-   -   (1) The primary sequence of a peptide segment of predetermined        length is scanned, and all hydrophobic aliphatic and aromatic        side chains present are identified. (2)The hydrophobic aliphatic        side chains are assigned a value greater than that for the        aromatic side chains; preferably about twice the value assigned        to the aromatic side chains, e.g., a value of 2 for a        hydrophobic aliphatic side chain and a value of 1 for an        aromatic side chain. (3) The values determined to be present are        summed for each overlapping amino acid residue segment (window)        of predetermined uniform length within the peptide, and the        total value for a particular segment (window) is assigned to a        single amino acid residue at an intermediate position of the        segment (window), preferably to a residue at about the midpoint        of the sampled segment (window). This procedure is repeated for        each sampled overlapping amino acid residue segment (window).        Thus, each amino acid residue of the peptide is assigned a value        that relates to the likelihood of a T-cell epitope being present        in that particular segment (window). (4) The values calculated        and assigned as described in Step 3, above, can be plotted        against the amino acid coordinates of the entire amino acid        residue sequence being assessed. (5) All portions of the        sequence which have a score of a predetermined value, e.g., a        value of 1, are deemed likely to contain a T-cell epitope and        can be modified, if desired.

This particular aspect of the present invention provides a generalmethod by which the regions of peptides likely to contain T-cellepitopes can be described. Modifications to the peptide in these regionshave the potential to modify the MHC Class II binding characteristics.

According to another aspect of the present invention, T-cell epitopescan be predicted with greater accuracy by the use of a moresophisticated computational method which takes into account theinteractions of peptides with models of MHC Class II alleles. Thecomputational prediction of T-cell epitopes present within a peptideaccording to this particular aspect contemplates the construction ofmodels of at least 42 MHC Class II alleles based upon the structures ofall known MHC Class II molecules and a method for the use of thesemodels in the computational identification of T-cell epitopes, theconstruction of libraries of peptide backbones for each model in orderto allow for the known variability in relative peptide backbone alphacarbon (Cα) positions, the construction of libraries of amino-acid sidechain conformations for each backbone dock with each model for each ofthe 20 amino-acid alternatives at positions critical for the interactionbetween peptide and MHC Class II molecule, and the use of theselibraries of backbones and side-chain conformations in conjunction witha scoring function to select the optimum backbone and side-chainconformation for a particular peptide docked with a particular MHC ClassII molecule and the derivation of a binding score from this interaction.

Models of MHC Class II molecules can be derived via homology modelingfrom a number of similar structures found in the Brookhaven Protein DataBank (“PDB”). These may be made by the use of semi-automatic homologymodeling software (Modeller, Sali A. & Blundell TL., 1993. J. Mol Biol234:779-815) which incorporates a simulated annealing function, inconjunction with the CHARMm force-field for energy minimisation(available from Molecular Simulations Inc., San Diego, Calif.).Alternative modeling methods can be utilized as well.

The present method differs significantly from other computationalmethods which use libraries of experimentally derived binding data ofeach amino-acid alternative at each position in the binding groove for asmall set of MHC Class II molecules (Marshall, K. W., et al., Biomed.Pept. Proteins Nucleic Acids, 1(3):157-162) (1995) or yet othercomputational methods which use similar experimental binding data inorder to define the binding characteristics of particular types ofbinding pockets within the groove, again using a relatively small subsetof MHC Class II molecules, and then ‘mixing and matching’ pocket typesfrom this pocket library to artificially create further ‘virtual’ MHCClass II molecules (Sturniolo T., et al., Nat. Biotech, 17(6): 555-561(1999). Both prior methods suffer the major disadvantage that, due tothe complexity of the assays and the need to synthesize large numbers ofpeptide variants, only a small number of MHC Class II molecules can beexperimentally scanned. Therefore the first prior method can only makepredictions for a small number of MHC Class II molecules. The secondprior method also makes the assumption that a pocket lined with similaramino-acids in one molecule will have the same binding characteristicswhen in the context of a different Class II allele and suffers furtherdisadvantages in that only those MHC Class II molecules can be‘virtually’ created which contain pockets contained within the pocketlibrary. Using the modeling approach described herein, the structure ofany number and type of MHC Class II molecules can be deduced, thereforealleles can be specifically selected to be representative of the globalpopulation. In addition, the number of MHC Class II molecules scannedcan be increased by making further models further than having togenerate additional data via complex experimentation. The use of abackbone library allows for variation in the positions of the Cα atomsof the various peptides being scanned when docked with particular MHCClass II molecules. This is again in contrast to the alternative priorcomputational methods described above which rely on the use ofsimplified peptide backbones for scanning amino-acid binding inparticular pockets. These simplified backbones are not likely to berepresentative of backbone conformations found in ‘real’ peptidesleading to inaccuracies in prediction of peptide binding. The presentbackbone library is created by superposing the backbones of all peptidesbound to MHC Class II molecules found within the Protein Data Bank andnoting the root mean square (RMS) deviation between the Cα atoms of eachof the eleven amino-acids located within the binding groove. While thislibrary can be derived from a small number of suitable available mouseand human structures (currently 13), in order to allow for thepossibility of even greater variability, the RMS figure for each C″-αposition is increased by 50%. The average Cα position of each amino-acidis then determined and a sphere drawn around this point whose radiusequals the RMS deviation at that position plus 50%. This sphererepresents all allowed Cα positions. Working from the Cα with the leastRMS deviation (that of the amino-acid in Pocket 1 as mentioned above,equivalent to Position 2 of the 11 residues in the binding groove), thesphere is three-dimensionally gridded, and each vertex within the gridis then used as a possible location for a Cα of that amino-acid. Thesubsequent amide plane, corresponding to the peptide bond to thesubsequent amino-acid is grafted onto each of these Cαs and the φ and ψangles are rotated step-wise at set intervals in order to position thesubsequent Cα. If the subsequent Cα falls within the ‘sphere of allowedpositions’ for this Cα than the orientation of the dipeptide isaccepted, whereas if it falls outside the sphere then the dipeptide isrejected.

This process is then repeated for each of the subsequent Cα positions,such that the peptide grows from the Pocket 1 Cα ‘seed’, until all ninesubsequent Cαs have been positioned from all possible permutations ofthe preceding Cαs. The process is then repeated once more for the singleCα preceding pocket 1 to create a library of backbone Cα positionslocated within the binding groove.

The number of backbones generated is dependent upon several factors: Thesize of the ‘spheres of allowed positions’; the fineness of the griddingof the ‘primary sphere’ at the Pocket 1 position; the fineness of thestep-wise rotation of the φ and ψ angles used to position subsequentCαs. Using this process, a large library of backbones can be created.The larger the backbone library, the more likely it will be that theoptimum fit will be found for a particular peptide within the bindinggroove of an MHC Class II molecule. Inasmuch as all backbones will notbe suitable for docking with all the models of MHC Class II moleculesdue to clashes with amino-acids of the binding domains, for each allelea subset of the library is created comprising backbones which can beaccommodated by that allele.

The use of the backbone library, in conjunction with the models of MHCClass II molecules creates an exhaustive database consisting of allowedside chain conformations for each amino-acid in each position of thebinding groove for each MHC Class II molecule docked with each allowedbackbone. This data set is generated using a simple steric overlapfunction where a MHC Class II molecule is docked with a backbone and anamino-acid side chain is grafted onto the backbone at the desiredposition. Each of the rotatable bonds of the side chain is rotatedstep-wise at set intervals and the resultant positions of the atomsdependent upon that bond noted. The interaction of the atom with atomsof side-chains of the binding groove is noted and positions are eitheraccepted or rejected according to the following criteria: The sum totalof the overlap of all atoms so far positioned must not exceed apre-determined value. Thus the stringency of the conformational searchis a function of the interval used in the step-wise rotation of the bondand the pre-determined limit for the total overlap. This latter valuecan be small if it is known that a particular pocket is rigid, howeverthe stringency can be relaxed if the positions of pocket side-chains areknown to be relatively flexible. Thus allowances can be made to imitatevariations in flexibility within pockets of the binding groove. Thisconformational search is then repeated for every amino-acid at everyposition of each backbone when docked with each of the MHC Class IImolecules to create the exhaustive database of side-chain conformations.

A suitable mathematical expression is used to estimate the energy ofbinding between models of MHC Class II molecules in conjunction withpeptide ligand conformations which have to be empirically derived byscanning the large database of backbone/side-chain conformationsdescribed above. Thus a protein is scanned for potential T-cell epitopesby subjecting each possible peptide of length varying between 9 and 20amino-acids (although the length is kept constant for each scan) to thefollowing computations: An MHC Class II molecule is selected togetherwith a peptide backbone allowed for that molecule and the side-chainscorresponding to the desired peptide sequence are grafted on. Atomidentity and interatomic distance data relating to a particularside-chain at a particular position on the backbone are collected foreach allowed conformation of that amino-acid (obtained from the databasedescribed above). This is repeated for each side-chain along thebackbone and peptide scores derived using a scoring function. The bestscore for that backbone is retained and the process repeated for eachallowed backbone for the selected model. The scores from all allowedbackbones are compared and the highest score is deemed to be the peptidescore for the desired peptide in that MHC Class II model. This processis then repeated for each model with every possible peptide derived fromthe protein being scanned, and the scores for peptides versus models aredisplayed.

In the context of the present invention, each ligand presented for thebinding affinity calculation is an amino-acid segment selected from apeptide or protein as discussed above. Thus, the ligand is a selectedstretch of amino acids about 9 to 20 amino acids in length derived froma peptide, polypeptide or protein of known sequence. The terms “aminoacids” and “residues” are hereinafter regarded as equivalent terms.

The ligand, in the form of the consecutive amino acids of the peptide tobe examined grafted onto a backbone from the backbone library, ispositioned in the binding cleft of an MHC Class II molecule from the MHCClass II molecule model library via the coordinates of the C″-α atoms ofthe peptide backbone and an allowed conformation for each side-chain isselected from the database of allowed conformations. The relevant atomidentities and interatomic distances are also retrieved from thisdatabase and used to calculate the peptide binding score. Ligands with ahigh binding affinity for the MHC Class II binding pocket are flagged ascandidates for site-directed mutagenesis. Amino-acid substitutions aremade in the flagged ligand (and hence in the protein of interest) whichis then retested using the scoring function in order to determinechanges which reduce the binding affinity below a predeterminedthreshold value. These changes can then be incorporated into the proteinof interest to remove T-cell epitopes.

Binding between the peptide ligand and the binding groove of MHC ClassII molecules involves non-covalent interactions including, but notlimited to: hydrogen bonds, electrostatic interactions, hydrophobic(lipophilic) interactions and Van der Walls interactions. These areincluded in the peptide scoring function as described in detail below.

It should be understood that a hydrogen bond is a non-covalent bondwhich can be formed between polar or charged groups and consists of ahydrogen atom shared by two other atoms. The hydrogen of the hydrogendonor has a positive charge where the hydrogen acceptor has a partialnegative charge. For the purposes of peptide/protein interactions,hydrogen bond donors may be either nitrogens with hydrogen attached orhydrogens attached to oxygen or nitrogen. Hydrogen bond acceptor atomsmay be oxygens not attached to hydrogen, nitrogens with no hydrogensattached and one or two connections, or sulphurs with only oneconnection. Certain atoms, such as oxygens attached to hydrogens orimine nitrogens (e.g. C═NH may be both hydrogen acceptors or donors.Hydrogen bond energies range from 3 to 7 Kcal/mol and are much strongerthan Van der Waal's bonds, but weaker than covalent bonds. Hydrogenbonds are also highly directional and are at their strongest when thedonor atom, hydrogen atom and acceptor atom are co-linear.

Electrostatic bonds are formed between oppositely charged ion pairs andthe strength of the interaction is inversely proportional to the squareof the distance between the atoms according to Coulomb's law. Theoptimal distance between ion pairs is about 2.8 Å. In protein/peptideinteractions, electrostatic bonds may be formed between arginine,histidine or lysine and aspartate or glutamate. The strength of the bondwill depend upon the pKa of the ionizing group and the dielectricconstant of the medium although they are approximately similar instrength to hydrogen bonds.

Lipophilic interactions are favorable hydrophobic-hydrophobic contactsthat occur between he protein and peptide ligand. Usually, these willoccur between hydrophobic amino acid side chains of the peptide buriedwithin the pockets of the binding groove such that they are not exposedto solvent. Exposure of the hydrophobic residues to solvent is highlyunfavorable since the surrounding solvent molecules are forced tohydrogen bond with each other forming cage-like clathrate structures.The resultant decrease in entropy is highly unfavorable. Lipophilicatoms may be sulphurs which are neither polar nor hydrogen acceptors andcarbon atoms which are not polar.

Van der Waal's bonds are non-specific forces found between atoms whichare 3-4 Å apart. They are weaker and less specific than hydrogen andelectrostatic bonds. The distribution of electronic charge around anatom changes with time and, at any instant, the charge distribution isnot symmetric. This transient asymmetry in electronic charge induces asimilar asymmetry in neighboring atoms. The resultant attractive forcesbetween atoms reaches a maximum at the Van der Waal's contact distancebut diminishes very rapidly at about 1 Å to about 2 Å. Conversely, asatoms become separated by less than the contact distance, increasinglystrong repulsive forces become dominant as the outer electron clouds ofthe atoms overlap. Although the attractive forces are relatively weakcompared to electrostatic and hydrogen bonds (about 0.6 Kcal/mol), therepulsive forces in particular may be very important in determiningwhether a peptide ligand may bind successfully to a protein.

In one embodiment, the Böhm scoring function (SCORE1 approach) is usedto estimate the binding constant. (Böhm, H. J., J. Comput Aided Mol.Des., 8(3):243-256 (1994) which is hereby incorporated in its entirety).In another embodiment, the scoring function (SCORE2 approach) is used toestimate the binding affinities as an indicator of a ligand containing aT-cell epitope (Böhm, H. J., J. Comput Aided Mol. Des., 12(4):309-323(1998) which is hereby incorporated in its entirety). However, the Böhmscoring functions as described in the above references are used toestimate the binding affinity of a ligand to a protein where it isalready known that the ligand successfully binds to the protein and theprotein/ligand complex has had its structure solved, the solvedstructure being present in the Protein Data Bank (“PDB”). Therefore, thescoring function has been developed with the benefit of known positivebinding data. In order to allow for discrimination between positive andnegative binders, a repulsion term must be added to the equation. Inaddition, a more satisfactory estimate of binding energy is achieved bycomputing the lipophilic interactions in a pairwise manner rather thanusing the area based energy term of the above Böhm functions.

Therefore, in a preferred embodiment, the binding energy is estimatedusing a modified Böhm scoring function. In the modified Böhm scoringfunction, the binding energy between protein and ligand (ΔG_(bind)) isestimated considering the following parameters: The reduction of bindingenergy due to the overall loss of translational and rotational entropyof the ligand (ΔG₀); contributions from ideal hydrogen bonds (ΔG_(hb))where at least one partner is neutral; contributions from unperturbedionic interactions (ΔG_(ionic)); lipophilic interactions betweenlipophilic ligand atoms and lipophilic acceptor atoms (ΔG_(lipo)); theloss of binding energy due to the freezing of internal degrees offreedom in the ligand, i.e., the freedom of rotation about each C—C bondis reduced (ΔG_(rot)); the energy of the interaction between the proteinand ligand (E_(VdW)). Consideration of these terms gives eguation 1:(ΔG_(bind))=(ΔG₀)+(ΔG_(hb)×N_(hb))+(ΔG_(ionic)×N_(ionic))+(ΔG_(lipo)×N_(lipo))+(ΔG_(rot)+N_(rot))+E_(VdW)).Where N is the number of qualifying interactions for a specific termand, in one embodiment, ΔG₀, ΔG_(hb), ΔG_(ionic), ΔG_(lipo) and ΔG_(rot)are constants which are given the values: 5.4, −4.7, −4.7, −0.17, and1.4, respectively.

The term N_(hb) is calculated according to equation 2:N _(hb)=Σ_(h-bonds) f(ΔR, Δα)×f(N _(neighb))×f _(pcs)f(ΔR, Δα) is a penalty function which accounts for large deviations ofhydrogen bonds from ideality and is calculated according to equation 3:f(ΔR, Δ−α)=f 1(ΔR)×f 2(Δα)Where:

-   -   f1(ΔR)=1 if ΔR<=TOL    -   or =1−(ΔR−TOL)/0.4 if ΔR<=0.4+TOL    -   or =0 if ΔR>0.4+TOL        And:    -   f2(Δα)=1 if Δα<30°    -   or =1−(Δα−30)/50 if Δα<=80°    -   or =0 if Δα>80°

-   TOL is the tolerated deviation in hydrogen bond length=0.25 Å

-   ΔR is the deviation of the H—O/N hydrogen bond length from the ideal    value=1.9 Å

-   Δα is the deviation of the hydrogen bond angle ∠_(N/O—H.,O/N) from    its idealized value of 180°

f(N_(neighb)) distinguishes between concave and convex parts of aprotein surface and therefore assigns greater weight to polarinteractions found in pockets rather than those found at the proteinsurface. This function is calculated according to equation 4 below:f(N_(neighb))=(N_(neighb)/N_(neighb,0))^(α where α=)0.5

-   -   N_(neighb) is the number of non-hydrogen protein atoms that are        closer than 5 Å to any given protein atom.    -   N_(neighb,0) is a constant=25    -   f_(pcs) is a function which allows for the polar contact surface        area per hydrogen bond and therefore distinguishes between        strong and weak hydrogen bonds and its value is determined        according to the following criteria:        f _(pcs)=β when A _(polar) /N _(HB)<10 Å²        or f _(pcs)=1 when A _(polar) /N _(HB)>10 Å²    -   A_(polar) is the size of the polar protein-ligand contact        surface    -   N_(HB) is the number of hydrogen bonds    -   β is a constant whose value=1.2

For the implementation of the modified Böhm scoring function, thecontributions from ionic interactions, ΔG_(ionic), are computed in asimilar fashion to those from hydrogen bonds described above since thesame geometry dependency is assumed.

The term N_(lipo) is calculated according to equation 5 below:N _(lipo)=Σ_(1L) f(r _(1L)[t1])f(r_(1L)) is calculated for all lipophilic ligand atoms, 1, and alllipophilic protein atoms, L, according to the following criteria:f(r_(1L))=1 when r_(1L) <=R 1 f(r_(1L))=(r_(1L) −R 1)/(R 2−R 1) when R2<r_(1L) >R1f(r_(1L))=0 when r_(1L) >=R2

-   -   Where: R1=r₁ ^(vdw)+r_(L) ^(vdw)+0.5    -   and R2=R1+3.0    -   and r₁ ^(vdw) is the Van der Waal's radius of atom 1    -   and r_(L) ^(vdw) is the Van der Waal's radius of atom L

The term N_(rot) is the number of rotable bonds of the amino acid sidechain and is taken to be the number of acyclic sp³sp³ and sp³-sp² bonds.Rotations of terminal —CH₃ or —NH₃ are not taken into account.

The final term, E_(VdW), is calculated according to equation 6 below:E _(VdW)=ε₁ε₂((r₁ ^(vdw)+r₂ ^(vdw)) ¹²/r¹²−(r₁ ^(vdw)+r₂ ^(vdw)) ⁶/r⁶),where:

-   -   ε₁ and ε₂ are constants dependant upon atom identity    -   r₁ ^(vdw)+r₂ ^(vdw) are the Van der Waal's atomic radii    -   r is the distance between a pair of atoms.

With regard to Equation 6, in one embodiment, the constants ε₁ and ε₂are given the atom values: C: 0.245, N: 0.283, O: 0.316, S: 0.316,respectively (i.e. for atoms of Carbon, Nitrogen, Oxygen and Sulphur,respectively). With regards to equations 5 and 6, the Van der Waal'sradii are given the atom values C: 1.85, N: 1.75, O: 1.60, S: 2.00 Å.

It should be understood that all predetermined values and constantsgiven in the equations above are determined within the constraints ofcurrent understandings of protein ligand interactions with particularregard to the type of computation being undertaken herein. Therefore, itis possible that, as this scoring function is refined further, thesevalues and constants may change hence any suitable numerical value whichgives the desired results in terms of estimating the binding energy of aprotein to a ligand may be used and hence fall within the scope of thepresent invention.

As described above, the scoring function is applied to data extractedfrom the database of side-chain conformations, atom identities, andinteratomic distances. For the purposes of the present description, thenumber of MHC Class II molecules included in this database is 42 modelsplus four solved structures. It should be apparent from the abovedescriptions that the modular nature of the construction of thecomputational method of the present invention means that new models cansimply be added and scanned with the peptide backbone library andside-chain conformational search function to create additional data setswhich can be processed by the peptide scoring function as describedabove. This allows for the repertoire of scanned MHC Class II moleculesto easily be increased, or structures and associated data to be replacedif data are available to create more accurate models of the existingalleles.

The present prediction method can be calibrated against a data setcomprising a large number of peptides whose affinity for various MHCClass II molecules has previously been experimentally determined. Bycomparison of calculated versus experimental data, a cut of value can bedetermined above which it is known that all experimentally determinedT-cell epitopes are correctly predicted.

It should be understood that, although the above scoring function isrelatively simple compared to some sophisticated methodologies that areavailable, the calculations are performed extremely rapidly. It shouldalso be understood that the objective is not to calculate the truebinding energy per se for each peptide docked in the binding groove of aselected MHC Class II protein. The underlying objective is to obtaincomparative binding energy data as an aid to predicting the location ofT-cell epitopes based on the primary structure (i.e. amino acidsequence) of a selected protein. A relatively high binding energy or abinding energy above a selected threshold value would suggest thepresence of a T-cell epitope in the ligand. The ligand may then besubjected to at least one round of amino-acid substitution and thebinding energy recalculated. Due to the rapid nature of thecalculations, these manipulations of the peptide sequence can beperformed interactively within the program's user interface oncost-effectively available computer hardware. Major investment incomputer hardware is thus not required. It would be apparent to oneskilled in the art that other available software could be used for thesame purposes. In particular, more sophisticated software which iscapable of docking ligands into protein binding-sites may be used inconjunction with energy minimization. Examples of docking software are:DOCK (Kuntz et al., J. Mol. Biol., 161:269-288 (1982)), LUDI (Böhm, H.J., J. Comput Aided Mol. Des., 8:623-632 (1994)) and FLEXX (Rarey M., etal., ISMB, 3:300-308 (1995)). Examples of molecular modeling andmanipulation software include: AMBER (Tripos) and CHARMm (MolecularSimulations Inc.). The use of these computational methods would severelylimit the throughput of the method of this invention due to the lengthsof processing time required to make the necessary calculations. However,it is feasible that such methods could be used as a ‘secondary screen’to obtain more accurate calculations of binding energy for peptideswhich are found to be ‘positive binders’ via the method of the presentinvention.

The limitation of processing time for sophisticated molecular mechanicor molecular dynamic calculations is one which is defined both by thedesign of the software which makes these calculations and the currenttechnology limitations of computer hardware. It may be anticipated that,in the future, with the writing of more efficient code and thecontinuing increases in speed of computer processors, it may becomefeasible to make such calculations within a more manageable time-frame.

Further information on energy functions applied to macromolecules andconsideration of the various interactions that take place within afolded protein structure can be found in: Brooks, B. R., et al., J.Comput. Chem., 4:187-217 (1983) and further information concerninggeneral protein-ligand interactions can be found in: Dauber-Osguthorpeet al., Proteins4(1):3 1-47(1988), which are incorporated herein byreference in their entirety. Useful background information can also befound, for example, in Fasman, G. D., ed., Prediction of ProteinStructure and the Principles of Protein Conformation, Plenum Press, NewYork, ISBN: 0-306 4313-9.

EXAMPLE 2 Method for Naïve T-cell Assay Using Synthetic Peptides

The interaction between MHC, peptide and T-cell receptor (TCR) providesthe structural basis for the antigen specificity of T-cell recognition.T-cell proliferation assays test the binding of peptides to MHC and therecognition of MHC/peptide complexes by the TCR. In vitro T-cellproliferation assays of the present example, involve the stimulation ofperipheral blood mononuclear cells (PBMCs), containing antigenpresenting cells (APCs) and T-cells. Stimulation is conducted in vitrousing synthetic peptide antigens, and in some experiments whole proteinantigen. Stimulated T-cell proliferation is measured using ³H-thymidine(³H-Thy) and the presence of incorporated ³H-Thy assessed usingscintillation counting of washed fixed cells.

Buffy coats from human blood stored for less than 12 hours are obtainedfrom the National Blood Service (Addenbrooks Hospital, Cambridge, UK).Ficoll-paque is obtained from Amersham Pharmacia Biotech (Amersham, UK).Serum free AIM V media for the culture of primary human lymphocytes andcontaining L-glutamine, 50 μg/ml streptomycin, 10 μg/ml gentomycin and0.1% human serum albumin is from Gibco-BRL (Paisley, UK). Syntheticpeptides are obtained from Pepscan (The Netherlands) and BabrahamTechnix (Cambridge, UK).

Erythrocytes and leukocytes are separated from plasma and platelets bygentle centrifugation of buffy coats. The top phase (containing plasmaand platelets) are removed and discarded. Erythrocytes and leukocytesare diluted 1:1 in phosphate buffered saline (PBS) and layered onto 15ml ficoll-paque (Amersham Pharmacia, Amersham UK). Centrifugation isdone according to the manufacturers recommended conditions and PBMCsharvested from the serum+PBS/ficoll paque interface. PBMCs are mixedwith PBS (1:1) and collected by centrifugation. The supernatant isremoved and discarded and the PBMC pellet resuspended in 50 ml PBS.Cells are again pelleted by centrifugation and the PBS supernatantdiscarded. Cells are resuspended using 50 ml AIM V media and at thispoint counted and viability assessed using trypan blue dye exclusion.Cells are again collected by centrifugation and the supernatantdiscarded. Cells are resuspended for cryogenic storage at a density of3×10⁷ per ml. The storage medium is 90% (v/v) heat inactivated AB humanserum (Sigma, Poole, UK) and 10% (v/v) DMSO (Sigma, Poole, UK). Cellsare transferred to a regulated freezing container (Sigma) and placed at−70° C. overnight before transferring to liquid N₂ for long termstorage. When required for use, cells are thawed rapidly in a water bathat 37° C. before transferring to 10 ml pre-warmed AIM V medium.

PBMC are stimulated with protein and peptide antigens in a 96 well flatbottom plate at a density of 2×10⁵ PBMC per well. PBMC are incubated for7 days at 37° C. before pulsing with ³H-Thy (Amersham-Phamacia,Amersham, UK). For the present study, synthetic peptides (15 mers) whichadvance by 3 amino acid increments are generated that span the entiresequence of hGH or all or any of peptides from Table 1 or peptidescontaining substitutions detailed in Table 2 or Table 3 can be generatedand used.

Each peptide is screened individually in triplicate against PBMC'sisolated from 20 naïve donors. Two control peptides that have previouslybeen shown to be immunogenic and a potent non-recall antigen KLH areused in each donor assay. The control antigens are as below: PeptideSequence C-32 Biotin-PKYVKQNTLKLAT Flu haemagglutinin 307-319 C-49KVVDQIKKISKPVQH Chlamydia HSP 60 peptide KLH Whole protein from KeyholeLimpet Hemocyanin.

Peptides are dissolved in DMSO to a final concentration of 10 mM, thesestock solutions were then diluted 1/500 in AIM V media (finalconcentration 20 μM). Peptides were added to a flat bottom 96 well plateto give a final concentration of 2 and 20 μM in a 100 μl. The viabilityof thawed PBMC's was assessed by trypan blue dye exclusion, cells werethen resuspended at a density of 2×10⁶ cells/ml, and 100 μl (2×10⁵PBMC/well) was transferred to each well containing peptides. Triplicatewell cultures are assayed at each peptide concentration. Plates areincubated for 7 days in a humidified atmosphere of 5% CO₂ at 37° C.Cells are pulsed for 18-21 hours with 1 μCi ³H-Thy/well beforeharvesting onto filter mats. CPM values are determined using a Wallacmicroplate beta top plate counter (Perkin Elmer) or similar. Results areexpressed as stimulation indices, determined using the followingformula:Proliferation to test peptide CPMProliferation in untreated wells CPM

For a naïve T-cell assay of this kind, a stimulation index of greaterthan 2.0 is taken as a positive score. Where the same test peptideachieves a stimulation index of greater than 2.0 in more than on donorsample this is taken as evidence of likely a immunodominant epitope.

1. A modified molecule having the biological activity of human growthhormone (hGH) and being substantially non-immunogenic or lessimmunogenic than any non-modified molecule having the same biologicalactivity when used in vivo.
 2. A molecule according to claim 1, whereinsaid loss of immunogenicity is achieved by removing one or more T-cellepitopes derived from the originally non-modified molecule.
 3. Amolecule according to claim 1, wherein said loss of immunogenicity isachieved by reduction in numbers of MHC allotypes able to bind peptidesderived from said molecule. 4-30. (cancelled).
 31. An isolated proteinthat is homologous to human growth hormone, the human growth hormonehaving an amino acid sequence (SEQ ID NO: 1) that includes at least oneT-cell epitope; the protein having substantially the same amino acidsequence as SEQ ID NO: 1, but including at least one less T-cellepitope; wherein the protein has substantially the same biologicalactivity as human growth hormone, but is less immunogenic than saidhuman growth hormone when both are exposed to the immune system of thesame species.
 32. The protein of claim 31 wherein the amino acidsequence of the protein includes one less T-cell epitope.
 33. Theprotein of claim 31 wherein the amino acid sequence of the proteindiffers from SEQ ID NO: 1 by one to nine amino acid residues.
 34. Theprotein of claim 31 wherein the amino acid sequence of the protein hasat least one less amino acid residue than SEQ ID NO:
 1. 35. The proteinof claim 31 wherein the amino acid sequence of the protein has at leastone more amino acid residue than SEQ ID NO:
 1. 36. The protein of claim31 wherein the amino acid sequence of the protein has the same number ofamino acid residues as SEQ ID NO:
 1. 37. The protein of claim 36 whereinthe amino acid sequence of the protein differs from SEQ ID NO: 1 by oneto nine amino acid residues.
 38. The protein of claim 31 wherein theamino acid sequence of the protein contains at least one amino acidsubstitution in SEQ ID NO: 1 selected from the group of amino acidsubstitutions set forth in Table
 2. 39. The protein of claim 38 whereinthe amino acid sequence of the protein further contains at least oneamino acid substitution in SEQ ID NO: 1 selected from the group of aminoacid substitutions set forth in Table
 3. 40. An isolated polypeptidehaving an amino acid sequence consisting of at least nine consecutiveamino acid residues of a sequence selected from the group of sequencesset forth in Table
 1. 41. An isolated polypeptide having an amino acidsequence selected from the group of sequences set forth in Table
 1. 42.An isolated polynucleotide encoding a protein of claim
 31. 43. Anisolated polynucleotide encoding a protein of claim
 38. 44. An isolatedpolynucleotide encoding a protein of claim
 39. 45. An isolatedpolynucleotide encoding a polypeptide of claim
 41. 46. A method ofpreparing a protein of claim 31, the method comprising the steps of: (i)identifying one or more potential T-cell epitopes within the amino acidsequence of human growth hormone (SEQ ID NO: 1); (ii) selecting at leastone sequence variant of at least one potential T-cell epitope identifiedin step (i) that eliminates or substantially reduces the MHC class IIbinding activity of the potential T-cell epitope; wherein the amino acidsequence of the selected variant differs from the amino acid sequence ofthe T-cell epitope identified in step (i) by at least one amino acidresidue; (iii) preparing, by recombinant DNA techniques, at least oneprotein that includes at least one variant selected in step (ii); (iv)evaluating the biological activity and immunogenicity of at least oneprotein prepared in step (iii); and (v) selecting a protein evaluated instep (iv) that has substantially the same biological activity as, butsubstantially less immunogenicity than human hormone.
 47. The method ofclaim 46 wherein step (i) is carried out by determining the MHC class IIbinding affinity of potential T-cell epitope segments of human growthhormone using an in vitro assay, an in silico technique, or a biologicalassay.
 48. The method of claim 46 wherein step (i) is carried out by:(a) selecting a region of the amino acid sequence of human growthhormone (SEQ ID NO: 1); (b) sequentially sampling overlapping amino acidresidue segments of predetermined uniform size and including at leastthree amino acid residues from the selected region; (c) calculating theMHC class II molecule binding score for each of the sampled segments bysumming assigned values for each hydrophobic amino acid residue sidechain present in the sampled amino acid residue segment; and (d)identifying at least one segment that is suitable for modification basedon the calculated MHC class II binding score for that segment to reducethe overall MHC class II binding score for the protein relative to thebinding score for human growth hormone.
 49. The method of claim 48wherein step (c) is carried out by using a Böhm scoring functionmodified to include a van der Waal's ligand-protein energy repulsiveterm and a ligand conformational energy term by: (1) selecting a modelfrom a first database consisting of MHC class II molecule models; (2)selecting an allowed peptide backbone from a second database consistingof allowed peptide backbones for the MHC class II molecule models instep (1); (3) identifying amino acid residue side chains present in eachsampled segment; (4) determining the binding affinity value for all sidechains present in each sampled segment; and (5) repeating each of steps(1) through (4) for each model in the first database and for eachbackbone in the second database.
 50. The method of claim 46 wherein step(ii) is carried out by substitution, addition, or deletion of one tonine amino acid residues from a potential T-cell epitope identified instep (i).
 51. The protein of claim 31 having an amino acid sequence thatis free from T-cell epitopes.
 52. A protein prepared by the method ofclaim
 46. 53. A pharmaceutical composition comprising a protein of claim31 and a pharmaceutically acceptable carrier therefor.
 54. Apharmaceutical composition comprising a protein of claim 38 and apharmaceutically acceptable carrier therefor.
 55. A pharmaceuticalcomposition comprising a protein of claim 39 and a pharmaceuticallyacceptable carrier therefor.
 56. A pharmaceutical composition comprisinga protein of claim 51 and a pharmaceutically acceptable carriertherefor.
 57. A pharmaceutical composition comprising a protein of claim52 and a pharmaceutically acceptable carrier therefor.